Streptavidin, a protein produced by Streptomyces avidinii, forms a very strong and specific non-covalent complex with the water soluble vitamin biotin. Streptavidin is a tetrameric protein that binds biotin with an affinity that is amongst the highest displayed for non-covalent interactions between a ligand and protein, with an association constant (Ka) estimated to be in the range of 10.sup.13 M.sup.-1 to 10.sup.15 M.sup.-1. This binding affinity is strong enough to be essentially irreversible under normal physiological solution conditions, and provides the basis for streptavidin and biotin's usefulness in a wide variety of clinical and industrial applications. See, Green, Adv. Prot. Chem. 29: 85-143 (1975).
Both streptavidin and the homologous protein avidin, which shares its high affinity for biotin, have been studied as paradigms of strong ligand-protein interactions. The X-ray crystal structures of streptavidin and avidin, both in their apo and holo forms, have been described. The sequences of both have also been reported, as have the construction of several streptavidin fusion proteins (Sano and Cantor, Biochem. Biophys. Res. Commun. 176: 571-577 (1991); U.S. Pat. No. 4,839,293). The structure-function origins of the unusually high affinity, however, have yet to be elucidated.
The streptavidin molecule displays a number of the common recognition motifs that have been identified for protein-ligand binding interactions. These include van der Waals dispersive attractions, which are largely mediated by the aromatic side chains of tryptophan residues, hydrogen bonding networks mediated by donor/acceptor side chains, and disorder-to-order transitions mediated by the ordering of surface polypeptide loops upon ligand binding.
Miyamoto and Kollman have reported a computational study that emphasizes the importance of hydrophobic/van der Waals dispersive attraction between ligand and protein. Miyamoto and Kollman, Proteins 16: 226-245 (1993) and Proc. Natl. Acad. Sci. USA 90: 8402-8406 (1993). These studies suggest that hydrophobic/van der Waals interactions contribute .sup..about. 18 kcal/mol to the absolute free energy of binding, while the electrostatic energy term (which includes hydrogen bonding interactions) contributes only .sup..about. 3 kcal/mol.
In addition to the extremely high binding affinity, the usefulness of streptavidin also arises from the unique architectural properties of the protein. Streptavidin is a tetramer of four identical subunits, with each subunit contributing a binding site for biotin. Because the tetramer has approximate two-fold symmetry, the binding sites are positioned in pairs on opposite sides of the molecule, making the protein an efficient molecular adaptor. This structural feature, along with the high affinity of streptavidin for biotin, has made the protein an important component in many technologies.
While the streptavidin tetramer displays nearly ideal 222 point group symmetry, there are two distinct protein--protein interfaces within the tetramer. Hendrickson et al., Proc. Natl. Acad. Sci. USA 86: 2190-2194 (1989); Weber et al., Science 243: 85-88 (1989). The first interface lies between two monomers that are related by the two-fold symmetry axis, and is defined by an extensive overlap of .beta.-barrel surfaces with complementary curvatures. This interface is characterized by a number of van der Waals, hydrogen bonding, and salt-bridge interactions. The close association of subunits at this interface defines the streptavidin dimer, with biotin binding sites related by the two-fold symmetry axis. The second tetramer interface defines the surface between pairs of these closely associated dimers (streptavidin is well described as a "dimer of dimers"). The dimer/dimer interface is characterized by a very loose "waistline" with minimal bonding interactions mediated largely by the C-terminal .beta.-strand 8 of the monomers. Thus, the dimer interface is structurally extensive while the dimer/dimer interface is structurally minimal. Despite the apparent lack of strong bonding interactions at the dimer/dimer interface, the streptavidin tetramer is exceedingly stable in both the biotin-free and biotin-bound states. The tetramer does not dissociate into smaller subunits in either 6 M urea or 6 M guanidinium hydrochloride. Kurzban et al., J. Biol. Chem. 266: 14470-14477 (1991).
Streptavidin and avidin are key components in four technological areas of great significance: 1) bioseparations/cell sorting; 2) imaging; 3) drug delivery; and 4) diagnostics (Wilchek and Bayer, in Meths. Enzymol. 184: 5-45 (1990)). In the separations area, these proteins have been used extensively in important cell sorting applications, where for example they are used to remove contaminating cells from hematopoietic stem cells prior to marrow transplantation. Berenson et al., Prog. Clin. Biol. Res. 377: 449-459 (1992). They have found similar wide use in cancer diagnostics, where they are used extensively in both research and clinical settings to test for the presence of various tumor specific biomarkers.
The imaging and drug delivery applications of streptavidin/avidin and biotin arise from the capability for simultaneous targeting and delivery of imaging agents or therapeutics to tumor cells. There is particularly significant emerging interest in the use of streptavidin/avidin for targeted delivery of imaging agents and therapeutics in vivo. Streptavidin/avidin has been used to deliver drugs, toxins and imaging agents to targeted cells both in vitro and in vivo. See, e.g., Meyer et al., Exp. Hematol. 19: 710-713 (1991). In these systems, streptavidin plays the crucial role of molecular adaptor between an antibody that serves as the targeting component, and a biotinylated therapeutic or imaging agent. With some strategies, cells are pre-targeted with the antibody-streptavidin conjugate, with subsequent delivery of the biotinylated agent. In other applications, a biotinylated antibody is first used to pre-target cells, with subsequent delivery of the streptavidin-biotinylated agent conjugate. A three-step delivery is also possible, using biotinylated antibody followed by streptavidin and then the biotinylated agent.
While streptavidin and avidin are incredibly useful molecules, they have important limitations, such as the inflexibility of four identical subunits having binding sites with extremely high affinity. Further, it has not been feasible to control the distribution of the subunits within the tetramer if the degeneracy of the subunits is removed (e.g., subunits with different affinities, subunits labeled with different imaging agents, subunits labeled with different drugs).
What is needed in the art is the ability to tailor the functional properties of individual subunits, and their geometrical distribution within the tetramer. This can be accomplished by manipulating important streptavidin structure-function relationships. A library of streptavidin mutants spanning a range of affinities and off- and on-rates for biotin and its derivatives would improve upon existing biotechnological applications for this already widely used system and open it to important new uses. Similarly, the ability to precisely define the subunit components and geometry will dramatically improve existing applications and provide new tools for cell separations, imaging, therapeutics and a variety of other technologies. Quite surprisingly, the present invention fulfills these and other related needs.